The signal transduction pathways driving morphogenesis and migration are still not well understood, yet defects in these processes underlie a wide spectrum of human diseases and syndromes, including mental retardation, chronic obstructive pulmonary disease (COPD), and cancer. The long term objectives of this proposal are to delineate the cellular mechanisms that control morphogenesis and migration in mammalian epithelial cells. The approaches used here involve the analysis of: (i) collective epithelial cell migration and (ii) the establishment of cell-cell junctions and apical/basal polarity in epithelial cells. The key hypothesis is that despite being very different biological processes, morphogenesis and migration share many of the same molecular components and signaling pathways and that Rho GTPases play a central role in both. In work funded by the current grant, we identified a number of regulators of Rho signaling pathways required for the collective migration of a human bronchial epithelial cell line, 16HBE, and developed time-lapse ex vivo imaging to show that Rac1 is required for the collective migration of anterior visceral endoderm (AVE) cells in embryonic day 5.5 mouse embryos. In Aim 1, micro-patterned surfaces will be used to identify the contribution of Rho pathways and polarity proteins to the collective behavior of migrating 16HBE cells, and microinjection techniques coupled with real time imaging will be used to identify molecular players in the collective migration of AVE cells in e5.5 mouse embryos. In other work funded by the current grant, we identified three kinases downstream of Rho and Cdc42 required for cell-cell junction assembly in 16HBE cells, and identified several additional Rho family target proteins required for 3D morphogenesis (polarized cyst formation) of the colorectal cell line Caco-2. In Aim 2, we will identify the kinase substrates involved in junction assembly in 16HBE cells, identify the molecular basis of apical/basal polarity establishment during the first cell division of Caco-2 cells grown in 3D, and delineate the contribution of Rho family targets to Caco-2 morphogenesis. This program of research will lead to a significant advance in understanding the control of migration and morphogenesis and provide greater insight into the underlying cell biology associated with a wide range of human disorders.